5’ and 3’ untranslated regions strongly enhance performance of geminiviral replicons in Nicotiana benthamiana leaves

We previously reported a recombinant protein production system based on a geminivirus replicon that yields high levels of vaccine antigens and monoclonal antibodies in plants. The bean yellow dwarf virus (BeYDV) replicon generates massive amounts of DNA copies, which engage the plant transcription machinery. However, we noticed a disparity between transcript level and protein production, suggesting that mRNAs could be more efficiently utilized. In this study, we systematically evaluated genetic elements from human, viral, and plant sources for their potential to improve the BeYDV system. The tobacco extensin terminator enhanced transcript accumulation and protein production compared to other commonly used terminators, indicating that efficient transcript processing plays an important role in recombinant protein production. Evaluation of human-derived 5’ untranslated regions (UTRs) indicated that many provided high levels of protein production, supporting their cross-kingdom function. Among the viral 5’ UTRs tested, we found the greatest enhancement with the tobacco mosaic virus omega leader. An analysis of the 5’ UTRs from the Arabidopsis thaliana and Nicotinana benthamiana photosystem I K genes found that they were highly active when truncated to include only the near upstream region, providing a dramatic enhancement of transgene production that exceeded that of the tobacco mosaic virus omega leader. The tobacco Rb7 matrix attachment region (MAR) inserted downstream from the gene of interest provided significant enhancement, which was correlated with a reduction in plant cell death. Evaluation of agrobacterium strains found that EHA105 enhanced protein production and reduced cell death compared to LBA4301 and GV3101. We used these improvements to produce Norwalk virus capsid protein at <20% total soluble protein, corresponding to 1.8mg/g leaf fresh weight, more than twice the highest level ever reported in a plant system. We also produced the monoclonal antibody rituximab at 1mg/g leaf fresh weight. Ausführliche Beschreibung

Gespeichert in:

Autor*in:	Andrew G. Diamos [verfasserIn] Sun Hee Rosenthal [verfasserIn] Hugh S Mason [verfasserIn]

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2016

Schlagwörter:	3' Untranslated Regions 5' Untranslated Regions Monoclonal antibody Geminivirus Nicotiana benthamiana virus-like particle

Übergeordnetes Werk:	In: Frontiers in Plant Science - Frontiers Media S.A., 2011, 7(2016)
Übergeordnetes Werk:	volume:7 ; year:2016

Links:	Link aufrufen Link aufrufen Link aufrufen Journal toc

DOI / URN:	10.3389/fpls.2016.00200

Katalog-ID:	DOAJ059732687

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allfields	10.3389/fpls.2016.00200 doi (DE-627)DOAJ059732687 (DE-599)DOAJe1d1f75a55b74ef0881b6ed0cd1c7434 DE-627 ger DE-627 rakwb eng SB1-1110 Andrew G. Diamos verfasserin aut 5’ and 3’ untranslated regions strongly enhance performance of geminiviral replicons in Nicotiana benthamiana leaves 2016 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier We previously reported a recombinant protein production system based on a geminivirus replicon that yields high levels of vaccine antigens and monoclonal antibodies in plants. The bean yellow dwarf virus (BeYDV) replicon generates massive amounts of DNA copies, which engage the plant transcription machinery. However, we noticed a disparity between transcript level and protein production, suggesting that mRNAs could be more efficiently utilized. In this study, we systematically evaluated genetic elements from human, viral, and plant sources for their potential to improve the BeYDV system. The tobacco extensin terminator enhanced transcript accumulation and protein production compared to other commonly used terminators, indicating that efficient transcript processing plays an important role in recombinant protein production. Evaluation of human-derived 5’ untranslated regions (UTRs) indicated that many provided high levels of protein production, supporting their cross-kingdom function. Among the viral 5’ UTRs tested, we found the greatest enhancement with the tobacco mosaic virus omega leader. An analysis of the 5’ UTRs from the Arabidopsis thaliana and Nicotinana benthamiana photosystem I K genes found that they were highly active when truncated to include only the near upstream region, providing a dramatic enhancement of transgene production that exceeded that of the tobacco mosaic virus omega leader. The tobacco Rb7 matrix attachment region (MAR) inserted downstream from the gene of interest provided significant enhancement, which was correlated with a reduction in plant cell death. Evaluation of agrobacterium strains found that EHA105 enhanced protein production and reduced cell death compared to LBA4301 and GV3101. We used these improvements to produce Norwalk virus capsid protein at <20% total soluble protein, corresponding to 1.8mg/g leaf fresh weight, more than twice the highest level ever reported in a plant system. We also produced the monoclonal antibody rituximab at 1mg/g leaf fresh weight. 3' Untranslated Regions 5' Untranslated Regions Monoclonal antibody Geminivirus Nicotiana benthamiana virus-like particle Plant culture Sun Hee Rosenthal verfasserin aut Hugh S Mason verfasserin aut In Frontiers in Plant Science Frontiers Media S.A., 2011 7(2016) (DE-627)662359240 (DE-600)2613694-6 1664462X nnns volume:7 year:2016 https://doi.org/10.3389/fpls.2016.00200 kostenfrei https://doaj.org/article/e1d1f75a55b74ef0881b6ed0cd1c7434 kostenfrei http://journal.frontiersin.org/Journal/10.3389/fpls.2016.00200/full kostenfrei https://doaj.org/toc/1664-462X Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 7 2016
spelling	10.3389/fpls.2016.00200 doi (DE-627)DOAJ059732687 (DE-599)DOAJe1d1f75a55b74ef0881b6ed0cd1c7434 DE-627 ger DE-627 rakwb eng SB1-1110 Andrew G. Diamos verfasserin aut 5’ and 3’ untranslated regions strongly enhance performance of geminiviral replicons in Nicotiana benthamiana leaves 2016 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier We previously reported a recombinant protein production system based on a geminivirus replicon that yields high levels of vaccine antigens and monoclonal antibodies in plants. The bean yellow dwarf virus (BeYDV) replicon generates massive amounts of DNA copies, which engage the plant transcription machinery. However, we noticed a disparity between transcript level and protein production, suggesting that mRNAs could be more efficiently utilized. In this study, we systematically evaluated genetic elements from human, viral, and plant sources for their potential to improve the BeYDV system. The tobacco extensin terminator enhanced transcript accumulation and protein production compared to other commonly used terminators, indicating that efficient transcript processing plays an important role in recombinant protein production. Evaluation of human-derived 5’ untranslated regions (UTRs) indicated that many provided high levels of protein production, supporting their cross-kingdom function. Among the viral 5’ UTRs tested, we found the greatest enhancement with the tobacco mosaic virus omega leader. An analysis of the 5’ UTRs from the Arabidopsis thaliana and Nicotinana benthamiana photosystem I K genes found that they were highly active when truncated to include only the near upstream region, providing a dramatic enhancement of transgene production that exceeded that of the tobacco mosaic virus omega leader. The tobacco Rb7 matrix attachment region (MAR) inserted downstream from the gene of interest provided significant enhancement, which was correlated with a reduction in plant cell death. Evaluation of agrobacterium strains found that EHA105 enhanced protein production and reduced cell death compared to LBA4301 and GV3101. We used these improvements to produce Norwalk virus capsid protein at <20% total soluble protein, corresponding to 1.8mg/g leaf fresh weight, more than twice the highest level ever reported in a plant system. We also produced the monoclonal antibody rituximab at 1mg/g leaf fresh weight. 3' Untranslated Regions 5' Untranslated Regions Monoclonal antibody Geminivirus Nicotiana benthamiana virus-like particle Plant culture Sun Hee Rosenthal verfasserin aut Hugh S Mason verfasserin aut In Frontiers in Plant Science Frontiers Media S.A., 2011 7(2016) (DE-627)662359240 (DE-600)2613694-6 1664462X nnns volume:7 year:2016 https://doi.org/10.3389/fpls.2016.00200 kostenfrei https://doaj.org/article/e1d1f75a55b74ef0881b6ed0cd1c7434 kostenfrei http://journal.frontiersin.org/Journal/10.3389/fpls.2016.00200/full kostenfrei https://doaj.org/toc/1664-462X Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 7 2016
allfields_unstemmed	10.3389/fpls.2016.00200 doi (DE-627)DOAJ059732687 (DE-599)DOAJe1d1f75a55b74ef0881b6ed0cd1c7434 DE-627 ger DE-627 rakwb eng SB1-1110 Andrew G. Diamos verfasserin aut 5’ and 3’ untranslated regions strongly enhance performance of geminiviral replicons in Nicotiana benthamiana leaves 2016 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier We previously reported a recombinant protein production system based on a geminivirus replicon that yields high levels of vaccine antigens and monoclonal antibodies in plants. The bean yellow dwarf virus (BeYDV) replicon generates massive amounts of DNA copies, which engage the plant transcription machinery. However, we noticed a disparity between transcript level and protein production, suggesting that mRNAs could be more efficiently utilized. In this study, we systematically evaluated genetic elements from human, viral, and plant sources for their potential to improve the BeYDV system. The tobacco extensin terminator enhanced transcript accumulation and protein production compared to other commonly used terminators, indicating that efficient transcript processing plays an important role in recombinant protein production. Evaluation of human-derived 5’ untranslated regions (UTRs) indicated that many provided high levels of protein production, supporting their cross-kingdom function. Among the viral 5’ UTRs tested, we found the greatest enhancement with the tobacco mosaic virus omega leader. An analysis of the 5’ UTRs from the Arabidopsis thaliana and Nicotinana benthamiana photosystem I K genes found that they were highly active when truncated to include only the near upstream region, providing a dramatic enhancement of transgene production that exceeded that of the tobacco mosaic virus omega leader. The tobacco Rb7 matrix attachment region (MAR) inserted downstream from the gene of interest provided significant enhancement, which was correlated with a reduction in plant cell death. Evaluation of agrobacterium strains found that EHA105 enhanced protein production and reduced cell death compared to LBA4301 and GV3101. We used these improvements to produce Norwalk virus capsid protein at <20% total soluble protein, corresponding to 1.8mg/g leaf fresh weight, more than twice the highest level ever reported in a plant system. We also produced the monoclonal antibody rituximab at 1mg/g leaf fresh weight. 3' Untranslated Regions 5' Untranslated Regions Monoclonal antibody Geminivirus Nicotiana benthamiana virus-like particle Plant culture Sun Hee Rosenthal verfasserin aut Hugh S Mason verfasserin aut In Frontiers in Plant Science Frontiers Media S.A., 2011 7(2016) (DE-627)662359240 (DE-600)2613694-6 1664462X nnns volume:7 year:2016 https://doi.org/10.3389/fpls.2016.00200 kostenfrei https://doaj.org/article/e1d1f75a55b74ef0881b6ed0cd1c7434 kostenfrei http://journal.frontiersin.org/Journal/10.3389/fpls.2016.00200/full kostenfrei https://doaj.org/toc/1664-462X Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 7 2016
allfieldsGer	10.3389/fpls.2016.00200 doi (DE-627)DOAJ059732687 (DE-599)DOAJe1d1f75a55b74ef0881b6ed0cd1c7434 DE-627 ger DE-627 rakwb eng SB1-1110 Andrew G. Diamos verfasserin aut 5’ and 3’ untranslated regions strongly enhance performance of geminiviral replicons in Nicotiana benthamiana leaves 2016 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier We previously reported a recombinant protein production system based on a geminivirus replicon that yields high levels of vaccine antigens and monoclonal antibodies in plants. The bean yellow dwarf virus (BeYDV) replicon generates massive amounts of DNA copies, which engage the plant transcription machinery. However, we noticed a disparity between transcript level and protein production, suggesting that mRNAs could be more efficiently utilized. In this study, we systematically evaluated genetic elements from human, viral, and plant sources for their potential to improve the BeYDV system. The tobacco extensin terminator enhanced transcript accumulation and protein production compared to other commonly used terminators, indicating that efficient transcript processing plays an important role in recombinant protein production. Evaluation of human-derived 5’ untranslated regions (UTRs) indicated that many provided high levels of protein production, supporting their cross-kingdom function. Among the viral 5’ UTRs tested, we found the greatest enhancement with the tobacco mosaic virus omega leader. An analysis of the 5’ UTRs from the Arabidopsis thaliana and Nicotinana benthamiana photosystem I K genes found that they were highly active when truncated to include only the near upstream region, providing a dramatic enhancement of transgene production that exceeded that of the tobacco mosaic virus omega leader. The tobacco Rb7 matrix attachment region (MAR) inserted downstream from the gene of interest provided significant enhancement, which was correlated with a reduction in plant cell death. Evaluation of agrobacterium strains found that EHA105 enhanced protein production and reduced cell death compared to LBA4301 and GV3101. We used these improvements to produce Norwalk virus capsid protein at <20% total soluble protein, corresponding to 1.8mg/g leaf fresh weight, more than twice the highest level ever reported in a plant system. We also produced the monoclonal antibody rituximab at 1mg/g leaf fresh weight. 3' Untranslated Regions 5' Untranslated Regions Monoclonal antibody Geminivirus Nicotiana benthamiana virus-like particle Plant culture Sun Hee Rosenthal verfasserin aut Hugh S Mason verfasserin aut In Frontiers in Plant Science Frontiers Media S.A., 2011 7(2016) (DE-627)662359240 (DE-600)2613694-6 1664462X nnns volume:7 year:2016 https://doi.org/10.3389/fpls.2016.00200 kostenfrei https://doaj.org/article/e1d1f75a55b74ef0881b6ed0cd1c7434 kostenfrei http://journal.frontiersin.org/Journal/10.3389/fpls.2016.00200/full kostenfrei https://doaj.org/toc/1664-462X Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 7 2016
allfieldsSound	10.3389/fpls.2016.00200 doi (DE-627)DOAJ059732687 (DE-599)DOAJe1d1f75a55b74ef0881b6ed0cd1c7434 DE-627 ger DE-627 rakwb eng SB1-1110 Andrew G. Diamos verfasserin aut 5’ and 3’ untranslated regions strongly enhance performance of geminiviral replicons in Nicotiana benthamiana leaves 2016 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier We previously reported a recombinant protein production system based on a geminivirus replicon that yields high levels of vaccine antigens and monoclonal antibodies in plants. The bean yellow dwarf virus (BeYDV) replicon generates massive amounts of DNA copies, which engage the plant transcription machinery. However, we noticed a disparity between transcript level and protein production, suggesting that mRNAs could be more efficiently utilized. In this study, we systematically evaluated genetic elements from human, viral, and plant sources for their potential to improve the BeYDV system. The tobacco extensin terminator enhanced transcript accumulation and protein production compared to other commonly used terminators, indicating that efficient transcript processing plays an important role in recombinant protein production. Evaluation of human-derived 5’ untranslated regions (UTRs) indicated that many provided high levels of protein production, supporting their cross-kingdom function. Among the viral 5’ UTRs tested, we found the greatest enhancement with the tobacco mosaic virus omega leader. An analysis of the 5’ UTRs from the Arabidopsis thaliana and Nicotinana benthamiana photosystem I K genes found that they were highly active when truncated to include only the near upstream region, providing a dramatic enhancement of transgene production that exceeded that of the tobacco mosaic virus omega leader. The tobacco Rb7 matrix attachment region (MAR) inserted downstream from the gene of interest provided significant enhancement, which was correlated with a reduction in plant cell death. Evaluation of agrobacterium strains found that EHA105 enhanced protein production and reduced cell death compared to LBA4301 and GV3101. We used these improvements to produce Norwalk virus capsid protein at <20% total soluble protein, corresponding to 1.8mg/g leaf fresh weight, more than twice the highest level ever reported in a plant system. We also produced the monoclonal antibody rituximab at 1mg/g leaf fresh weight. 3' Untranslated Regions 5' Untranslated Regions Monoclonal antibody Geminivirus Nicotiana benthamiana virus-like particle Plant culture Sun Hee Rosenthal verfasserin aut Hugh S Mason verfasserin aut In Frontiers in Plant Science Frontiers Media S.A., 2011 7(2016) (DE-627)662359240 (DE-600)2613694-6 1664462X nnns volume:7 year:2016 https://doi.org/10.3389/fpls.2016.00200 kostenfrei https://doaj.org/article/e1d1f75a55b74ef0881b6ed0cd1c7434 kostenfrei http://journal.frontiersin.org/Journal/10.3389/fpls.2016.00200/full kostenfrei https://doaj.org/toc/1664-462X Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 7 2016
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callnumber-first	S - Agriculture
author	Andrew G. Diamos
spellingShingle	Andrew G. Diamos misc SB1-1110 misc 3' Untranslated Regions misc 5' Untranslated Regions misc Monoclonal antibody misc Geminivirus misc Nicotiana benthamiana misc virus-like particle misc Plant culture 5’ and 3’ untranslated regions strongly enhance performance of geminiviral replicons in Nicotiana benthamiana leaves
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topic_title	SB1-1110 5’ and 3’ untranslated regions strongly enhance performance of geminiviral replicons in Nicotiana benthamiana leaves 3' Untranslated Regions 5' Untranslated Regions Monoclonal antibody Geminivirus Nicotiana benthamiana virus-like particle
topic	misc SB1-1110 misc 3' Untranslated Regions misc 5' Untranslated Regions misc Monoclonal antibody misc Geminivirus misc Nicotiana benthamiana misc virus-like particle misc Plant culture
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title	5’ and 3’ untranslated regions strongly enhance performance of geminiviral replicons in Nicotiana benthamiana leaves
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title_full	5’ and 3’ untranslated regions strongly enhance performance of geminiviral replicons in Nicotiana benthamiana leaves
author_sort	Andrew G. Diamos
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title_sort	5’ and 3’ untranslated regions strongly enhance performance of geminiviral replicons in nicotiana benthamiana leaves
callnumber	SB1-1110
title_auth	5’ and 3’ untranslated regions strongly enhance performance of geminiviral replicons in Nicotiana benthamiana leaves
abstract	We previously reported a recombinant protein production system based on a geminivirus replicon that yields high levels of vaccine antigens and monoclonal antibodies in plants. The bean yellow dwarf virus (BeYDV) replicon generates massive amounts of DNA copies, which engage the plant transcription machinery. However, we noticed a disparity between transcript level and protein production, suggesting that mRNAs could be more efficiently utilized. In this study, we systematically evaluated genetic elements from human, viral, and plant sources for their potential to improve the BeYDV system. The tobacco extensin terminator enhanced transcript accumulation and protein production compared to other commonly used terminators, indicating that efficient transcript processing plays an important role in recombinant protein production. Evaluation of human-derived 5’ untranslated regions (UTRs) indicated that many provided high levels of protein production, supporting their cross-kingdom function. Among the viral 5’ UTRs tested, we found the greatest enhancement with the tobacco mosaic virus omega leader. An analysis of the 5’ UTRs from the Arabidopsis thaliana and Nicotinana benthamiana photosystem I K genes found that they were highly active when truncated to include only the near upstream region, providing a dramatic enhancement of transgene production that exceeded that of the tobacco mosaic virus omega leader. The tobacco Rb7 matrix attachment region (MAR) inserted downstream from the gene of interest provided significant enhancement, which was correlated with a reduction in plant cell death. Evaluation of agrobacterium strains found that EHA105 enhanced protein production and reduced cell death compared to LBA4301 and GV3101. We used these improvements to produce Norwalk virus capsid protein at <20% total soluble protein, corresponding to 1.8mg/g leaf fresh weight, more than twice the highest level ever reported in a plant system. We also produced the monoclonal antibody rituximab at 1mg/g leaf fresh weight.
abstractGer	We previously reported a recombinant protein production system based on a geminivirus replicon that yields high levels of vaccine antigens and monoclonal antibodies in plants. The bean yellow dwarf virus (BeYDV) replicon generates massive amounts of DNA copies, which engage the plant transcription machinery. However, we noticed a disparity between transcript level and protein production, suggesting that mRNAs could be more efficiently utilized. In this study, we systematically evaluated genetic elements from human, viral, and plant sources for their potential to improve the BeYDV system. The tobacco extensin terminator enhanced transcript accumulation and protein production compared to other commonly used terminators, indicating that efficient transcript processing plays an important role in recombinant protein production. Evaluation of human-derived 5’ untranslated regions (UTRs) indicated that many provided high levels of protein production, supporting their cross-kingdom function. Among the viral 5’ UTRs tested, we found the greatest enhancement with the tobacco mosaic virus omega leader. An analysis of the 5’ UTRs from the Arabidopsis thaliana and Nicotinana benthamiana photosystem I K genes found that they were highly active when truncated to include only the near upstream region, providing a dramatic enhancement of transgene production that exceeded that of the tobacco mosaic virus omega leader. The tobacco Rb7 matrix attachment region (MAR) inserted downstream from the gene of interest provided significant enhancement, which was correlated with a reduction in plant cell death. Evaluation of agrobacterium strains found that EHA105 enhanced protein production and reduced cell death compared to LBA4301 and GV3101. We used these improvements to produce Norwalk virus capsid protein at <20% total soluble protein, corresponding to 1.8mg/g leaf fresh weight, more than twice the highest level ever reported in a plant system. We also produced the monoclonal antibody rituximab at 1mg/g leaf fresh weight.
abstract_unstemmed	We previously reported a recombinant protein production system based on a geminivirus replicon that yields high levels of vaccine antigens and monoclonal antibodies in plants. The bean yellow dwarf virus (BeYDV) replicon generates massive amounts of DNA copies, which engage the plant transcription machinery. However, we noticed a disparity between transcript level and protein production, suggesting that mRNAs could be more efficiently utilized. In this study, we systematically evaluated genetic elements from human, viral, and plant sources for their potential to improve the BeYDV system. The tobacco extensin terminator enhanced transcript accumulation and protein production compared to other commonly used terminators, indicating that efficient transcript processing plays an important role in recombinant protein production. Evaluation of human-derived 5’ untranslated regions (UTRs) indicated that many provided high levels of protein production, supporting their cross-kingdom function. Among the viral 5’ UTRs tested, we found the greatest enhancement with the tobacco mosaic virus omega leader. An analysis of the 5’ UTRs from the Arabidopsis thaliana and Nicotinana benthamiana photosystem I K genes found that they were highly active when truncated to include only the near upstream region, providing a dramatic enhancement of transgene production that exceeded that of the tobacco mosaic virus omega leader. The tobacco Rb7 matrix attachment region (MAR) inserted downstream from the gene of interest provided significant enhancement, which was correlated with a reduction in plant cell death. Evaluation of agrobacterium strains found that EHA105 enhanced protein production and reduced cell death compared to LBA4301 and GV3101. We used these improvements to produce Norwalk virus capsid protein at <20% total soluble protein, corresponding to 1.8mg/g leaf fresh weight, more than twice the highest level ever reported in a plant system. We also produced the monoclonal antibody rituximab at 1mg/g leaf fresh weight.
collection_details	GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700
title_short	5’ and 3’ untranslated regions strongly enhance performance of geminiviral replicons in Nicotiana benthamiana leaves
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An analysis of the 5’ UTRs from the Arabidopsis thaliana and Nicotinana benthamiana photosystem I K genes found that they were highly active when truncated to include only the near upstream region, providing a dramatic enhancement of transgene production that exceeded that of the tobacco mosaic virus omega leader. The tobacco Rb7 matrix attachment region (MAR) inserted downstream from the gene of interest provided significant enhancement, which was correlated with a reduction in plant cell death. Evaluation of agrobacterium strains found that EHA105 enhanced protein production and reduced cell death compared to LBA4301 and GV3101. We used these improvements to produce Norwalk virus capsid protein at <20% total soluble protein, corresponding to 1.8mg/g leaf fresh weight, more than twice the highest level ever reported in a plant system. 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5’ and 3’ untranslated regions strongly enhance performance of geminiviral replicons in Nicotiana benthamiana leaves

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